This invention relates to the identification and selection of genetically transformed cells. More specifically, this invention describes a vector-host cloning system in which genetic transformation with foreign nucleic acid is accompanied by a measurable change in surface binding properties of a host organism.
The ability to selectively recombine genetic material from different organisms in vitro and to cause the resulting "recombinant" material to be replicated and/or used to direct the expression of proteins in a host organism, has transformed the study of biology and greatly enhanced its practical utility, e.g., Cohen et al., U.S. Pat. No. 4,237,224. Applications of this "cloning" or "recombinant nucleic acid" technology include the expression of medically useful human proteins in bacterial cell culture, the creation of immortalized genetic "libraries" containing the entire genome of a particular organism, and the segregation and replication of foreign nucleic acid in preparation for nucleic acid sequencing.
Generally, the cloning process includes the following steps: (i) foreign nucleic acid fragments are prepared having the appropriate size and cohesive end regions, the foreign nucleic acid being either a digest of genomic nucleic acid or cDNA; (ii) a vector nucleic acid is cleaved with one or more restriction enzymes, preferably at a unique site, to form a vector nucleic acid having cohesive end regions suitable for binding to the cohesive end regions of the foreign nucleic acid fragments, where the vector nucleic acid includes (a) elements which allow the inserted foreign nucleic acid fragments to survive and autonomously replicate in a host organism and (b) a selectable marker in order to facilitate the recognition and isolation of cells carrying the foreign nucleic acid; (iii) the foreign nucleic acid fragments are contacted with the cleaved vector nucleic acid under conditions favoring combination of the foreign nucleic acid fragments and the vector nucleic acid, and the vector and foreign nucleic acid are ligated together, thereby forming a recombinant vector-foreign nucleic acid; (iv) the recombinant vector-foreign nucleic acid is introduced into the host organism, thereby genetically transforming the host organism; (v) the host organisms containing the recombinant vector-foreign nucleic acid are separated from untransformed cells and cells containing only parent vector nucleic acid, and optionally, (vi) the separated cells are subjected to a secondary screen for specific nucleic acid inserts, e.g., by contacting with nucleic acid probes or by contacting their expressed proteins with antibody probes.
A key step in the above cloning process is the identification and isolation of host organisms which have been successfully transformed with recombinant vector-foreign nucleic acid, i.e., step (v) above. A number of products can result from the reaction of the cleaved vector nucleic acid with the foreign nucleic acid fragments. The products of the recombination reaction are a heterogeneous mixture of recombinant vector-foreign nucleic acid molecules together with religated parental vector molecules. In fact, for many systems, the vast majority of cells are transformed with parental vector nucleic acid only, e.g., less than 0.1% of the host organisms are transformed with vector-foreign nucleic acid. This low frequency of vector-foreign nucleic acid transformation creates a large background of cells containing no foreign nucleic acid, thereby dramatically increasing the effort required to perform any secondary screening. The automation of step (v) is a particularly important issue for large-scale cloning projects where millions of potential clones must be screened, e.g., the construction of libraries for genomic sequencing projects.
There are four methods that are commonly used to identify host organisms that contain recombinant vector-foreign nucleic acid, including (i) restriction analysis of small-scale preparations of plasmid nucleic acid, (ii) .alpha.-complementation, (iii) insertional inactivation, and, (iv) screening by hybridization (see Molecular Cloning 2nd Ed., Sambrook et al, Chapter 1, Cold Spring Harbor Laboratory Press (1989)).
In the restriction analysis method, a number of independently transformed host organisms are picked from a parent culture and grown up in small-scale cultures. Plasmid nucleic acids are isolated from each culture and then analyzed by digestion with restriction enzymes followed by gel electrophoresis.
In the .alpha.-complementation method, the cloning vectors carry a short segment of Escherichia coli nucleic acid that contains the regulatory sequences and the coding information for the first 146 amino acids of the .beta.-galactosidase gene (lacZ). Embedded in this coding region is a polycloning site. These vectors are used in combination with host organisms that code for the carboxy-terminal portion of .beta.-galactosidase. While neither the host-encoded nor the vector-encoded fragments are themselves active, they can associate to form an enzymatically active protein. This type of complementation is called .alpha.-complementation. The Lac+ bacteria that result from .alpha.-complementation are easily recognized because they form blue colonies in the presence of the chromogenic substrate X-gal. However, insertion of a fragment of foreign nucleic acid into the polycloning site of the vector results in the production of an amino-terminal fragment that is not capable of .alpha.-complementation. Host organisms carrying recombinant vectors therefore form white colonies. The recombinant vectors can then be visually identified and manually "picked" from the culture.
The insertional inactivation method is used with vectors that carry two or more antibiotic resistance genes and an appropriate distribution of restriction enzyme cleavage sites. The foreign nucleic acid and the vector nucleic acid are digested with restriction enzymes that recognize sites located in only a first antibiotic resistance gene. After ligating the two nucleic acids, the ligation mixture is used to transform E. coli. Transformants are selected that are resistant to the second antibiotic. Some of the colonies that grow in the presence of the second antibiotic will contain recombinant vectors; others will contain parental vector nucleic acid that has religated during ligation without insertion of foreign nucleic acid. To discriminate between the two types of transformants, separate plates containing the first or second antibiotic are inoculated with a number of colonies in patches in identical locations. The colonies that grow in the presence of the first antibiotic contain plasmids with active resistance genes, and it is unlikely that such plasmids contain insertions of foreign nucleic acid. It is likely that the colonies that do not grow in the presence of the first antibiotic but do grow in the presence of the second antibiotic carry the foreign nucleic acid sequences.
In hybridization screening techniques, a replica of the host organism colonies is transferred to a filter support, the cells are lysed releasing their nucleic acid, and the nucleic acid is then probed with labeled sequence-specific nucleic acid probes. The location of probe-positive colonies is then used to identify the location of recombinant cells on the original culture plate.
The above methods for identifying cells which have been transformed with recombinant vectors each require a number of cumbersome steps resulting in significant barriers to intelligent automation. Even the most streamlined .alpha.-complementation method requires the analyst to (i) grow-up the putatively transformed cells in a culture plate, (ii) visually identify cells having a specified color, (iii) pick cells having a specified color, and (iv) transfer the picked cells to a secondary location. While automated systems for picking individual colonies have been demonstrated, they require complicated robotic and image processing apparatus, making them unsuitable for cost effective, routine application, e.g., Uber et al., Biotechniques 11: 642-648 (1991); Jones et al., Nucleic Acids Research 20: 4599-4606 (1992). Moreover, because the .alpha.-complementation approach requires cells to be plated out, it is not amenable to the selection of cells which can not be grown on culture plates, e.g., certain eucaryotic tissue cells.